Drug development efforts involve a continuum of activities initiated by target selection of a molecule. Since all drugs work at the level of the cell, those targets are usually proteins that somehow are involved in cellular communication pathways. Signal transduction pathways are key to normal cell function. Aberrations in the expression of intracellular molecules and coordinated interactions of signal transduction pathways are associated with a variety of diseases and, thus, are the focus of drug, discovery efforts. Phosphorylation of proteins in signal transduction pathways is one of the key covalent modifications that occur in multicellular organisms. The enzymes that carry out this modification are the protein kinases, which catalyze the transfer of the phosphate from ATP to tyrosine, serine or threonine residues on protein substrates. Phosphorylation of these amino acid residues can alter the function and/or location of the protein within the cell. This change can involve changes in the enzymatic activity of the affected protein and/or create binding sites for the recruitment of other signaling proteins. Because protein kinases are critical components of many cellular signaling pathways, their catalytic activity is often tightly regulated. Abnormalities in protein kinase activity result in different patterns of phosphorylation that can dramatically alter cell function. Indeed, many drug discovery efforts involve the identification of therapeutic agents that selectively suppress or augment protein kinase activity in order to treat a disease. This invention is designed to provide assays and reagents to monitor protein kinase activity.
The targeted residues for phosphorylation can be contained in a full-length, biologically active molecule of recombinant or natural origin. Most methods currently employed for measuring protein kinase activity use peptide substrates, which include the targeted phosphorylation residue. This art is taught in U.S. Pat. No. 6,066,462 (Quantitation of individual protein kinase activity) incorporated herein by reference. This method differs from the present invention in that the peptide substrate does not contain all possible phosphorylation sites that can be acted on by kinases and thus may not truly reflect activity on a natural protein. The invention described herein can be used with whole molecule or fragments, of natural or recombinant origin. Also, the delineation of activity at different phosphorylation sites requires in the invention, a different PSSA for detection as opposed to a different peptide in U.S. Pat. No. 6,066,462.
Another method for detection of kinase activity involves use of a generic antibody that binds to all phospho-tyrosine residues. This method is described in U.S. Pat. No. 5,766,863 (Kinase receptor activation assay) incorporated herein by reference. This method suffers from an inability to discriminate among phosphorylated tyrosine residues on a molecule. This method does not address detection of phospho-serine or phosphothreonine events since the anti-phospho-tyrosine antibody does not detect such phosphorylated residues. In contrast, the method described herein uses antibodies, which bind to the sequence specific residues surrounding the phosphorylated amino acid plus the phospho-residue itself. The reagents used in this invention are capable of detecting phosphorylated threonine, serine or tyrosine molecules.
The current invention and related methods are applicable to a wide range of signal transduction proteins (see Table I for a partial list). Three examples are illustrated below using important molecular targets of current interest in basic research and disease-oriented pharmaceutical study.
Currently, neurobiologists are focusing efforts on the proteins in the brain that can be associated with disease. One such protein is called Tau, a neuronal microtubule associated protein found predominantly in axons. The function of Tau is to promote tubulin polymerization and stabilize microtubules, but it also serves to link certain signaling pathways to the cytoskeleton. Tau phosphorylation regulates both normal and pathological functions of this protein. Tau, in its hyper-phosphorylated form, is the major component of paired helical filaments (PHF), the building block of neurofibrillary lesions that are often found in the brains of individuals with Alzheimer's disease (AD). Hyperphosphorylation impairs the microtubule binding function of Tau, resulting in the destabilization of microtubules in AD brains, ultimately leading to neuronal degeneration. Hyperphosphorylated Tau is also found in a range of other central nervous system disorders. Numerous serine/threonine kinases, including GSK-33, PKA, PKC, CDK5, MARK, INK, p38MAPK and casein kinase II, can phosphorylate Tau.
Detection of in vitro kinase activity is critical for screening compounds that may be able to inhibit this activity and therefore could be useful in ameliorating various neurodegenerative diseases where Tau phosphorylation is abnormally high. Current efforts exist to identify drugs that might suppress kinase activity towards the Tau protein; however, these methods suffer from poor sensitivity and low specificity. Phosphorylation at individual Serine or Threonine residues within the Tau protein has been shown to correlate with disease. This invention overcomes both of these deficiencies in the described ‘art’.
U.S. Pat. No. 5,601,985 relates to methods of detecting abnormally phosphory, lated Tau Protein; U.S. Pat. No. 5,843,779 relates to monoclonal antibodies directed against the microtubule-associated protein, Tau, and hybridomas secreting these antibodies; U.S. Pat. No. 5,733,734 relates to methods of screening for Alzheimer's disease or disease associated with the accumulation of paired helical filaments and U.S. Pat. No. 6,066,462 relates to quantitation of individual protein kinase activity. These patents are incorporated herein by reference.
In addition to the detection of Tau phosphorylation in AD, other models exist to show the general applicability of the currently described format for monitoring protein kinase activity. For the purposes of illustration, we have also designed assays around the intranuclear Retinoblastoma (Rb) protein important in cell cycle regulation and a cell surface receptor molecule (EGFR), which are both described in detail below.
Retinoblastoma protein (Rb), the tumor suppressor product of the retinoblastoma susceptibility gene, is a 110 kDa protein which plays an important role in regulating cell growth and differentiation. Loss of its function leads to uncontrolled cell growth and tumor development. Mutational inactivation of the Rb gene is found in all retinoblastomas and in a variety of other human malignancies including cancers of breast, lung, colon, prostate, osteosarcomas, soft tissue sarcomas, and leukemia. Central to the role of the Rb protein as a tumor suppressor is the ability of Rb to suppress inappropriate proliferation by arresting cells in the GI phase of the cell cycle. Rb protein exerts its growth suppressive function by binding to transcription factors including E2F-1, PU.1, ATF-2, UBF, Elf-1, and c-Abl. The binding of Rb protein is governed by its phosphorylation state. Hypo- or under-phosphorylated forms of Rb bind and sequester transcription factors, most notably those of the E2F/DP family, inhibiting the transcription of genes required to traverse the 01 to S phase boundary of the cell cycle. This cell cycle inhibitory function is abrogated when Rb undergoes phosphorylation catalyzed by specific complexes of cyclins and cyclin-dependent protein kinases (cdks).
Rb contains at least 16 consensus serine/threonine phosphorylation sites for cdks, although the significance of all these sites is still unclear. It has been demonstrated that phosphorylation of threonine 821 on Rh is catalyzed by cdk2/complex such as Cyclin E/cdk2 and Cyclin A/cdk2. The phosphorylation of threonine 821 disrupts the interaction of Rb with the proteins containing the sequence LXCXE, where L=leucine, C=cysteinc, E=glutamic acid, and X=any amino acid residue. The dephosphorylation of Rb protein returns Rb to its active, growth suppressive state. Removal of phosphates on Rb appears to be carried out by a multimeric complex of protein phosphatase type 1 (PP I) and noncatalytic regulatory subunits at the completion of mitosis. The quantitation of Rb phosphorylated at specific amino acid residues gives important information regarding the activity of kinases as well as the functional state of the Rb protein itself. For the purposes of illustration, we designed an assay to quantitate the amount of Rb protein that is specifically phosphorylated at threonine 821 using an ELISA format. This assay does not recognize Rb phosphorylated at sites other than [pT821] or when it is in the non-phosphorylated form. Samples can be controlled for Rb content by parallel measurement of total Rb protein.
WO 01/11367 (Assay of the phosphorylation activity of cyclin/CDK complex on retinoblastoma (RB) protein for identifying compounds which modify this activity) describes a method for detecting kinase activity by ELISA using a synthetic peptide and a monoclonal antibody that recognizes the phosphorylated form of the peptide. The basis of this method is the coating of a solid phase with a synthetic peptide containing the consensus sequence of a region upon which a kinase acts. The peptide is allowed to come in contact with a kinase that allows a specific residue on that peptide to become phosphorylated. The activity of the kinase then is estimated by the binding of the generic monoclonal antibody to the target phosphopeptide. Our invention differs from WO 01/11367 in that it uses a natural protein as the substrate for kinase activity. This feature is superior to the use of peptides since all naturally occurring phosphorylation sites would be present and the protein would be presented in its normal conformation. The use of a single monoclonal antibody recognizing phosphoserine (clone 2B9) also does not allow any discrimination of the many phosphorylation sites that naturally occur on Rb protein. Our use of specific PSSAs allows that distinction as well as the detection of phosphothreonine and phosphotyrosine residues allowing a profile of Rb phosphorylation sites to be constructed.
As a third example of the utility of this approach, a cell surface receptor was studied and a kinase-dependent ELISA designed. The Epidermal growth factor receptor (EGFR) belongs to the family of receptor tyrosine kinases (RTKs), which regulate cell growth, survival, proliferation and differentiation. EGFR is expressed at full length as a 170 kDa type I transmembrane glycoprotein which consists of an extracellular ligandbinding domain, a single hydrophobic transmembrane region, and an intracellular domain that exhibits tyrosine enzymatic activity and which is involved in signal transduction. Several deletions in the extra- and intracellular domain of the EGFR have been found in a number of tumors. For example, EGFRvill is a 145 kDa protein with a deletion of exons 2-7 in EGFR mRNA. A 100 kDa truncated EGFR without the cytoplasmic domain is observed in the culture supernatant from A431 cells, a human epidermal carcinoma cell line.
EGFR is activated by binding of a number of ligands such as EGF, transforming growth factor a (TGFα), amphiregulin, betacellulin, heparin binding EGF-like growth factor (HB-EGF) and epiregulin. The binding causes EGFR homo- and heterodimerization and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain, which involves rapid activation of its intrinsic tyrosine kinase activity. Phosphorylation of tyrosine residues in the COOH-terminal tail of the EGFR. serve as binding sites for cytosolic signaling proteins containing Src homology 2 (SH2) domains. Several sites of in vivo phosphorylation have been identified in the EGFR including Tyr845, Tyr992, Tyr1068, Tyr1086, and Tyr1173. These sites bind and activate a variety of downstream signaling proteins that contain SH2 domains, including growth factor receptor-binding protein 2 (Grb2), Src homology and collagen domain protein (She) and phospholipase C-γ (PLCγ). The binding of these or other signaling proteins to the receptor and/or their phosphorylation results in transmission of subsequent signaling events that culminate in DNA synthesis and cell division.
Elevated expression and/or amplification of the EGFR have been found in breast, bladder, glioma, colon, lung, squamous cell, head and neck, ovarian, and pancreatic cancers. Selective compounds have been developed that target either the extracellular ligand-binding domain of EGFR or the intracellular tyrosine kinase region, resulting in interference with the signaling pathways that modulate mitogenic and other cancer-promoting responses. These potential anticancer agents include a number of small molecule, tyrosine kinase inhibitors.